Ethanol is one of the most widely abused substances in the world, yet the molecular mechanisms of action of ethanol are poorly understood. Ethanol has been shown to disrupt ion channel functions in vitro through interactions with hydrophobic domains of channel subunit proteins and an understanding of the mechanisms that are likely to underlie some of the acute effects of ethanol has begun to emerge. It is still unclear, however, how many of the more complex behavioral responses to ethanol, such as tolerance and dependence, are mediated at a molecular level. We have pursued studies in C. elegans, as a model system, to understand the different molecular mechanisms of action of ethanol. Through genetic studies, we have identified proteins that are targets of ethanol or required for normal ethanol responses of C. elegans. In many cases, homologous proteins are also thought to be required for behavioral responses to ethanol in mammalian systems. We have demonstrated and characterized a form of acute tolerance in C. elegans that can be observed during sustained but brief ethanol exposures. After more prolonged exposure to ethanol and development of tolerance, wild type animals exhibit an intriguing change in their behavior that consists of an "ethanol-seeking" response, which we have quantified in a preference-type assay. This change is strengthened with longer ethanol exposures of several days. We have demonstrated that this response is partially dependent on dopaminergic and serotonergic function, as mutants with known defects in dopamine or serotonin synthesis exhibit substantially reduced ethanol-seeking. In addition, we have identified two novel mutants that are defective in the development of ethanol-seeking behavior. These ethanol-seeking defective mutants (esd-1 and esd-2) are also defective in the development of tolerance to ethanol. A subset of mutants defective in ethanol tolerance are found to be defective in ethanol seeking behavior, suggesting that the molecular mechanisms underlying the development of tolerance and ethanol-seeking behavior partially overlap. We now propose to complete the mapping of esd-1 and esd-2 and to clone and characterize the corresponding genes to gain insight into the molecular mechanisms that underlie this change in the desirability of ethanol over time. We will also pursue further detailed characterizations of the ethanol seeking response in wild type animals, and we will test additional mutants for defects in ethanol-seeking, as observed with esd-1 and esd-2. The determination of novel molecular mechanisms that underlie ethanol-seeking in C. elegans may enhance our understanding of changes in ethanol preference in vertebrate systems. PUBLIC HEALTH RELEVANCE: This project seeks to identify and characterize at a molecular level the mechanisms that underlie ethanol- seeking behavior in C. elegans or changes in the desirability of ethanol. Such mechanisms may be conserved. A greater understanding of the molecular pathways mediating such behavioral changes could lead to the development of medications to treat individuals who are alcohol dependent.